All neurotransmitters require an efficient mechanism to terminate their action. Water-soluble neurotransmitters diffuse unassisted in the cytosol to their respective catabolic enzyme for breakdown, after being transported into cells. The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are the major signaling molecules for the cannabinoid receptors CB1 and CB2. Unlike most other neurotransmitters, the endocannabinoids are neutral hydrophobic molecules. The hypothesis of this proposal is that, owing to their neutral lipid structure and their insolubility in cytosol, the inactivation of AEA and 2-AG occurs by a unique pathway. It is proposed that they bind to fatty acid binding proteins (FABPs) for intracellular trafficking to their inactivating enzymes. Accordingly, these studies will examine endogenous levels of FABPs in different cells. Furthermore, these studies will elucidate the role of FABPs upon the rate of endocannabinoid metabolism by assaying the cellular uptake and hydrolysis of AEA and 2-AG. The proof that FABPs are intracellular transporters of endocannabinoids will be demonstrated by measuring commensurate changes in metabolism after up-regulation of FABPs by transfection, down-regulation of FABPs by RNA interference or inactivation of FABPs by specific inhibitors. Herein we describe, for the first time, proteins that bind endocannabinoids and function as intracellular transporters. To further strengthen our confidence in the hypothesis that FABPs are intracellular transporters of endocannabinoids, experiments will also be conducted to show that two alternative mechanisms (simple diffusion or endocytosis) are less likely. From a health related viewpoint, understanding the mechanism of endocannabinoid inactivation and identifying FABPs as a new drug target drug target may lead to treatment for addiction, mood disorders, pain and inflammation, cognition, and appetite regulation. For example, competitive inhibitors of AEA and 2-AG binding to specific FABPs will decrease the hydrolysis of AEA and 2-AG as well as their cellular uptake, raising their levels at the synapse and producing a cannabinoid tone. PUBLIC HEALTH RELEVANCE: The two endocannabinoid neurotransmitters, anandamide and 2-AG, are required for normal physiological functioning in humans. Understanding how these compounds are inactivated in the body are the focus of this proposal and will lead to new targets for pharmaceutical drugs. Since anandamide and 2-AG signaling molecules are involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement;drugs that prevent their inactivation may be good for treating drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.